Inhibition and transcriptional silencing of a subtilisin-like proprotein convertase, PACE4/SPC4, reduces the branching morphogenesis of and AQP5 expression in rat embryonic submandibular gland

The submandibular gland (SMG) develops through the epithelial-mesenchymal interaction mediated by many growth/differentiation factors including activin and BMPs, which are synthesized as inactive precursors and activated by subtilisin-like proprotein convertases (SPC) following cleavage at their R-X-K/R-R site. Here, we found that Dec-RVKR-CMK, a potent inhibitor of SPC, inhibited the branching morphogenesis of the rat embryonic SMG, and caused low expression of a water channel AQP5, in an organ culture system. Dec-RVKR-CMK also decreased the expression of PACE4, a SPC member, but not furin, another SPC member, suggesting the involvement of PACE4 in the SMG development. Heparin, which is known to translocate PACE4 in the extracellular matrix into the medium, and an antibody specific for the catalytic domain of PACE4, both reduced the branching morphogenesis and AQP5 expression in the SMG. The inhibitory effects of Dec-RVKR-CMK were partially rescued by the addition of recombinant BMP2, whose precursor is one of the candidate substrates for PACE4 in vivo. Further, the suppression of PACE4 expression by siRNAs resulted in decreased expression of AQP5 and inhibition of the branching morphogenesis in the present organ culture system. These observations suggest that PACE4 regulates the SMG development via the activation of some growth/differentiation factors.     

Disruption of proprotein convertase 1/3 (PC1/3) expression in mice causes innate immune defects and uncontrolled cytokine secretion

The proprotein convertase 1/3 is expressed in the regulated secretory pathway of neural and endocrine cells. Its major function is in the post-translational processing and activation of precursor proteins. The PC1/3 knock-out (KO) mouse model has allowed us to elucidate its physiological functions in studies focused primarily on neuroendocrine tissues. However, PC1/3 is also expressed in cells of the immune system, mainly in macrophages. The present study explores the effects of innate immune challenge in the PC1/3 KO mouse. PC1/3 KO mice have an enlarged spleen with marked disorganization of the marginal zone and red pulp. Immunohistochemical studies using various markers demonstrate a depletion of dendritic cells in PC1/3 KO spleens. When challenged with lipopolysaccharide, PC1/3 KO mice are more susceptible to septic shock than wild-type controls or other PC KO mice, such as PC2 and PC7 null mice. Plasma levels of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) were very significantly elevated in PC1/3 KO mice, consistent with a hypercytokinemia, i.e. indicative of a major systemic uncontrolled inflammatory response or cytokine storm. Peritoneal macrophages isolated from PC1/3 KO mice also demonstrate elevated cytokine secretion when treated with LPS. Electron micrographs show morphological features indicating a prolonged activation of these cells following LPS stimulation. We also present evidence that the proinflammatory T(h)1 pathway is dominant in the PC1/3 KO mouse model. We conclude that aside from its important role in neuroendocrine functions PC1/3 also has an important role in the regulation of the innate immune system, most likely through the regulation of cytokine secretion in macrophages.     

Association of the rs6235 variant in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene with obesity and related traits in a Taiwanese population

One particularly interesting single nucleotide polymorphism (SNP), rs6235 (encoding an S690T substitution), in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene has been widely associated with obesity in several European cohorts. The present study was intended to investigate the association between the PCSK1 rs6235 SNP and the prevalence of overweight or obesity, or obesity-related metabolic traits in a Taiwanese population. A total of 964 Taiwanese subjects with general health examinations were analyzed. Our data revealed no association of PCSK1 rs6235 with the risk of obesity or overweight in the complete subjects. However, the PCSK1 rs6235 SNP exhibited a significant association with overweight among the male subjects (P = 0.03), but not among the female subjects. Furthermore, the carriers of GG variant had a significantly higher waist circumference than those with the CC variant (82.5 ± 11.5 vs. 81.2 ± 10.2 cm; P = 0.01) and those with the CG variant (82.5 ± 11.5 vs. 81.4 ± 10.4 cm; P = 0.021). In addition, the carriers of GG variant had a higher diastolic blood pressure than those with the CC variant (81.9 ± 14.2 vs. 80.3 ± 12.9 mm Hg; P = 0.023). Our study indicates that the PCSK1 rs6235 SNP may contribute to the risk of overweight in men and predict obesity-related metabolic traits such as waist circumference and diastolic blood pressure in Taiwanese subjects.     

LsGRP1, a class II glycine-rich protein of Lilium,confers plant resistance via mediating innate immune activation and inducing fungal programmed cell death

Defence-related LsGRP1 is a leaf-specific plant class II glycine-rich protein (GRP) involved in salicylic acid-induced systemic resistance against grey mould caused by necrotrophic Botrytis elliptica in lily (Lilium) cultivar Stargazer. The C-terminal region of LsGRP1 (LsGRP1^C) can inhibit fungal growth in vitro via a mechanism of inducing fungal apoptosis programmed cell death (PCD). In this study, the role of LsGRP1 in induced defence mechanism was investigated using LsGRP1-silenced Stargazer lily and LsGRP1-transgenic Arabidopsis thaliana. LsGRP1 silencing in lily was found to slightly inhibit plant growth and greatly increase the susceptibility to B. elliptica by suppressing callose deposition and early reactive oxygen species (ROS) accumulation. In contrast, LsGRP1-transgenic Arabidopsis showed higher resistance to Botrytis cinerea and also to Pseudomonas syringae pv. tomato DC3000 as compared to the wild type, accompanied with the enhancement of callose deposition and ROS accumulation. Additionally, LsGRP1 silencing increased plant cell death caused by B. elliptica secretion and reduced pathogen-associated molecular pattern (PAMP)-triggered defence activation in Stargazer lily. Consistently, LsGRP1 expression boosted PAMP-triggered defence responses and effector recognition-induced hypersensitive response in Arabidopsis. Moreover, fungal apoptosis PCD triggered by LsGRP1 in an LsGRP1^C-dependent manner was demonstrated by leaf infiltration with LsGRP1^C-containing recombinant proteins in Stargazer lily. Based on these results, we presume that LsGRP1 plays roles in plant defence via functioning as a pathogen-inducible switch for plant innate immune activation and acting as a fungal apoptosis PCD inducer to combat pathogen attack.     

A respiratory syncytial virus persistent-infected cell line system reveals the involvement of SOCS1 in the innate antiviral response

HEp-2 cells persistently infected with respiratory syncytial virus(RSV) are a heterogeneous mixture of viral antigen-positive and-negative variants; however, the mechanism through which viral replication becomes latent remains unclear. In this study, we investigated the potential mechanism by which RSV escapes from innate immune surveillance. Persistent-infected RSV HEp-2 cells were isolated and cell clones were passaged. The RSV-persistent cells produced viruses at a lower titer, resisted wild-type RSV re-infection, and secreted high levels of interferon-β(IFN-β), macrophage inflammatory protein-1α(Mip-1α), interleukin-8(IL-8), and Rantes. Toll-like receptor 3(TLR3), retinoic acid inducible gene-I(RIG-I), and suppressor of cytokine signaling 1(SOCS1) levels were upregulated in these cells. The silencing of TLR3 m RNA decreased the expression of SOCS1 protein and the secretion of cytokines. RSV-persistent cells are in an inflammatory state; upregulation of SOCS1 is related to the TLR3 signaling pathway, which could be associated with the mechanism of viral persistence.     

Partial restoration of T‐cell function in aged mice by in vitro blockade of the PD‐1/ PD‐L1 pathway

Programmed cell death‐1 (PD‐1) is a newly characterized negative regulator of immune responses. The interaction of PD‐1 with its ligands (PD‐L1 and PD‐L2) inhibits T‐cell proliferation and cytokine production in young mice. Increased PD‐1 expression has been described during chronic infections, inducing chronic activation of the immune system to control it. As aging is associated with chronic immune activation, PD‐1 may contribute to age‐associated T‐cell dysfunction. Our data showed the following results in aged mice: (i) the number of PD‐1‐expressing T cells and the level of expression of PD‐Ls was increased on dendritic cell subsets and T cells; (ii) PD‐1⁺ T cells were exhausted effector memory T cells, as shown by their lower level of CD127, CD25 and CD28, as well as their limited proliferative and cytokine‐producing capacity; (iii) the expression of PD‐1 was up‐regulated after T‐cell receptor‐mediated activation of CD8⁺ T cells, but not of CD4⁺ T cells; (iv) blockade of the PD‐1/PD‐L1 pathway moderately improved the cytokine production of T cells from old mice but did not restore their proliferation; and (v) blockade of the PD‐1/PD‐L1 pathway did not restore function of PD‐1⁺ T cells; its effect appeared to be exclusively mediated by increased functionality of the PD‐1^? T cells. Our data thus suggest that blockade of the PD‐1/PD‐L1 is not likely to be efficient at restoring exhausted T‐cell responses in aged hosts, although improving the responses of PD‐1^? T cells may prove to be a helpful strategy in enhancing primary responses.     

A single region of the Phytophthora infestans avirulence effector Avr3b functions in both cell death induction and plant immunity suppression

As a destructive plant pathogen, Phytophthora infestans secretes diverse host-entering RxLR effectors to facilitate infection. One critical RxLR effector, PiAvr3b, not only induces effector-triggered immunity (ETI), which is associated with the potato resistance protein StR3b, but also suppresses pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). To date, the molecular basis underlying such dual activities remains unknown. Based on phylogenetic analysis of global P. infestans isolates, we found two PiAvr3b isoforms that differ by three amino acids. Despite this sequence variation, the two isoforms retain the same properties in activating the StR3b-mediated hypersensitive response (HR) and inhibiting necrosis induced by three PAMPs (PiNpp, PiINF1, and PsXeg1) and an RxLR effector (Pi10232). Using a combined mutagenesis approach, we found that the dual activities of PiAvr3b were tightly linked and determined by 88 amino acids at the C-terminus. We further determined that either the W60 or the E134 residue of PiAvr3b was essential for triggering StR3b-associated HR and inhibiting PiNpp- and Pi10232-associated necrosis, while the S99 residue partially contributed to PTI suppression. Additionally, nuclear localization of PiAvr3b was required to stimulate HR and suppress PTI, but not to inhibit Pi10232-associated cell death. Our study revealed that PiAvr3b suppresses the plant immune response at different subcellular locations and provides an example in which a single amino acid of an RxLR effector links ETI induction and cell death suppression.     

Activation of protein phosphatase-2A1 by HIV-1 Vpr cell death causing peptide in intact CD(4+) T cells and in vitro

HIV-1, the etiologic agent of human AIDS, causes cell death in host and non-host cells via HIV-1 Vpr, one of its auxiliary gene product. HIV-1 Vpr can also cause cell cycle arrest in several cell types. The cellular processes that link HIV-1 Vpr to the cell death machinery are not well characterized. Here, we show that the C terminal portion of HIV-1 Vpr which encompasses amino acid residues 71-96 (HIV-1 Vpr(71-96)), also termed HIV-1 Vpr cell death causing peptide, is an activator of protein phosphatase-2A(1) when applied extracellularly to CD(4+) T cells. HIV-1 Vpr(71-96) is a direct activator of protein phosphatase-2A(1) that has been purified from CD(4+) T cells. Full length HIV-1 Vpr by itself does not cause the activation of protein phosphatase-2A(1) in vitro. HIV-1 Vpr(71-96) also causes the activation of protein phosphatase-2A(0) and protein phosphatase-2A(1) from brain, liver, and adipose tissues. These results indicate that HIV-1 can cause cell death of infected cells and non-infected host and non-host cells via HIV-1 Vpr derived C terminal peptide(s) which act(s) by cell penetration and targeting of a key controller of the cell death machinery, namely, protein phosphatase-2A(1). The activation of other members of the protein phosphatase-2A subfamily of enzymes which are involved in the control of several metabolic pathways in brain, liver, and adipose tissues by HIV-1 Vpr derived C terminal peptide(s) may underlie various metabolic disturbances that are associated with HIV-1 infection.     

AFAP1在博来霉素诱导的A549细胞衰老中的作用机制研究

目的:研究AFAP1在博来霉素诱导的A549细胞衰老模型中的作用及分子机制。方法:用50μg/m L的博来霉素处理A549细胞5天建立细胞衰老模型。用相同浓度的博来霉素处理细胞1-5天观察细胞从周期阻滞到衰老的过程,SA-β-Gal染色检测衰老细胞数目,用Western blot方法检测AFAP1、p21、c-Src等蛋白表达。过表达AFAP1后,观察细胞衰老状态及各蛋白表达水平变化。结果:50μg/m L的博来霉素处理A549细胞5天后可以建立细胞衰老模型,表现为BLM组SA-β-Gal阳性细胞数升高(P0.01)且细胞体积显著增大(P0.01),p21表达水平升高。在衰老的A549细胞中,AFAP1和激活型(Src p Y416)表达水平变化一致,从BLM处理后出现升高第4天开始明显下降在第5天最低,c-Src和Src p Y527表达水平不变。过表达AFAP1后再用博来霉素诱导,SA-β-Gal阳性细胞数及细胞体积、Src p Y416和p21表达与空载对照比较未发现有明显差异(P0.05)。结论:衰老的A549细胞中AFAP1表达下调,c-Src活性降低;过表达AFAP1不能减轻博来霉素诱导的A549细胞衰老,也不能抑制衰老细胞中的c-Src的活性下降。     

Design and synthesis of novel 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol- 2-yl)urea derivatives with potent anti-CML activity throughout PI3K/AKT signaling pathway

In this investigation, a series of 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4- thiadiazol-2-yl)urea receptor tyrosine kinase inhibitors were synthesized by a simple and efficient structure-based design. Structure-activity relationship (SAR) analysis of these compounds based on cellular assays led to the discovery of a number of compounds that showed potent activity against human chronic myeloid leukemia (CML) cell line K562, but very weak or no cellular toxicity through monitoring the growth kinetics of K562 cell during a period of 72 h using the real-time live-cell imaging. Among these compounds, 1-(5-((6-((3-morpholinopropyl) amino)pyrimidin-4-yl)thio)-1,3,4-thiadiazol-2-yl)-3-(4-(trifluoromethyl)phenyl)urea (7) exhibited the least cellular toxicity and better biological activity in cellular assays (K562, IC₅₀: 0.038 μM). Compound 7 also displayed very good induced-apoptosis effect for human CML cell line K562 and exerted its effect via a significantly reduced protein phosphorylation of PI3K/Akt signal pathway by Human phospho-kinase array analysis. In vitro results indicate that 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4- thiadiazol-2-yl)urea derivatives are lead molecules for further development as treatment of chronic myeloid leukemia and cancer.     

Leukotriene-C4 Synthase,a Critical Enzyme in the Activation of Store-independent Orai1/Orai3 Channels,Is Required for Neointimal Hyperplasia

Leukotriene-C₄ synthase (LTC₄S) generates LTC₄ from arachidonic acid metabolism. LTC₄ is a proinflammatory factor that acts on plasma membrane cysteinyl leukotriene receptors. Recently, however, we showed that LTC₄ was also a cytosolic second messenger that activated store-independent LTC₄-regulated Ca²⁺ (LRC) channels encoded by Orai1/Orai3 heteromultimers in vascular smooth muscle cells (VSMCs). We showed that Orai3 and LRC currents were up-regulated in medial and neointimal VSMCs after vascular injury and that Orai3 knockdown inhibited LRC currents and neointimal hyperplasia. However, the role of LTC₄S in neointima formation remains unknown. Here we show that LTC₄S knockdown inhibited LRC currents in VSMCs. We performed in vivo experiments where rat left carotid arteries were injured using balloon angioplasty to cause neointimal hyperplasia. Neointima formation was associated with up-regulation of LTC₄S protein expression in VSMCs. Inhibition of LTC₄S expression in injured carotids by lentiviral particles encoding shRNA inhibited neointima formation and inward and outward vessel remodeling. LRC current activation did not cause nuclear factor for activated T cells (NFAT) nuclear translocation in VSMCs. Surprisingly, knockdown of either LTC₄S or Orai3 yielded more robust and sustained Akt1 and Akt2 phosphorylation on Ser-473/Ser-474 upon serum stimulation. LTC₄S and Orai3 knockdown inhibited VSMC migration in vitro with no effect on proliferation. Akt activity was suppressed in neointimal and medial VSMCs from injured vessels at 2 weeks postinjury but was restored when the up-regulation of either LTC₄S or Orai3 was prevented by shRNA. We conclude that LTC₄S and Orai3 altered Akt signaling to promote VSMC migration and neointima formation.     

Immunohistochemical analysis of Bin1/Amphiphysin II in human tissues: diverse sites of nuclear expression and losses in prostate cancer

The Bin1/Amphiphysin II gene encodes at least seven alternately spliced adapter proteins that have been implicated in membrane dynamics and nuclear processes. Nuclear localized Bin1 polypeptides have tumor suppressor and proapoptotic activities, suggesting that Bin1 may suppress cancer in tissues where nuclear expression may occur. One question is the extent to which human tissues express nuclear Bin1 isoforms. A secondary issue has been the need for a specific antibody that can detect all the splice isoforms expressed by the human, mouse, and rat Bin1 genes. Using a novel mouse monoclonal antibody with these characteristics, we performed an immunohistochemical analysis of Bin1 expression in a panel of normal human tissues. We also compared the expression profile of Bin1 in normal or malignant tissues derived from human prostate, where Bin1 is a candidate tumor suppressor gene. In brain, a distinct nuclear staining pattern overlapped with a cytosolic staining pattern present in certain layers of the cerebral cortex and cerebellum. Bone marrow cells displayed mainly nuclear localization whereas peripheral lymphoid cells exhibited mainly cytosolic localization. In several epithelial tissues, nuclear or nucleocytosolic staining patterns were displayed by basal cells in skin, breast, or prostate, whereas cytosolic or plasma membrane-associated staining patterns were noted in gastrointestinal cells. Interestingly, a striking gradient of expression was observed in gastrointestinal epithelia, particularly in the large intestine, with the strongest staining displayed by cells destined to undergo apoptosis at the villus tip. In prostate, Bin1 staining was frequently absent in cases of primary prostate adenocarcinoma. This study used a novel reagent to document the extent of expression of nuclear Bin1 isoforms, which exhibit cancer suppression and proapoptotic activity in human cells.